Patching up a Broken Heart: Post-Infarct Cardiac Scar Digestion And Healthy Tissue Regeneration...
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Transcript of Patching up a Broken Heart: Post-Infarct Cardiac Scar Digestion And Healthy Tissue Regeneration...
Patching up a Broken Heart:Post-Infarct Cardiac Scar DigestionAnd Healthy Tissue Regeneration
20.020
May 14, 2008
Anna ShcherbinaDerek Ju
Prarthna DesaiAmber Lin
Aditya KohliRobbie Barbero
Michael Oh
We'd like to thank Professor Gredzinsky for his valuable advice
Impact of the Project
• All living organisms have scars of some sort (topical, internal, etc.)
•Topical scars are a commonplace imperfection with a variety of already-existing treatment options
• Scarring in the heart is a lot more perilous
•The formation of heart scars after heart attacks results in inefficiency of blood pumping and higher occurrences of arrhythmias
cardiac scar tissue(google.com)
Impact (continued)
• What if we could eliminate the tissue build-up that forms after heart attacks and re-form healthy tissue?
•Our project presents a new system of treating cardiac scars
•Giving someone back his heart tissue could, both literally and figuratively, give back his life
The General Idea:Binding
•Monoclonal antibodies are injected into the bloodstream. •One end binds to non-phosphorylated light myosin chains in the ECM of cardiac scar cells.•The other end binds to the engineered E. coli bacteria.
•Engineered bacteria are injected into the bloodstream and bind to the monoclonal antibody on the cardiac tissue.
The General Idea: Digesting and Regenerating
•After binding, the E. coli secrete collagenase•Collagenase digests collagen, the main component of scar tissue•A protein tether may be necessary for more targeted digestion
•At the same time, the E. coli secrete periostin•A growth factor that stimulates the formation of healthy cardiac tissue.
General Idea : Termination
•Collagenase and periostin function on a similar time scale and can thus be secreted simultaneously
• When the scar tissue has been digested, the E. coli no longer has a binding site
•The bacterium is unbound from the heart and carried away by the bloodstream
•Several injections may be necessary to ensure complete removal of scar tissue
Device Diagram
Scar DigestionDevice
Scar binding device
Trigger
RegeneratorDevice
Scar tissue moleculeScar digesting molecule
Heart regeneration molecule
Bacterial Cell
A
B
C
D
E
F
Regulatory Device
G
H
Timing Diagram
A
B
C
D
E
F
G
H
E. coli binds to scar
Trigger mechanism is fully activated
Scar has been fully digested
Bacteria cell
Scar binding device
Trigger
Scar digesting Device
Regenerator Device
Scar Tissue Molecule
Scar Digesting Molecule
Regenrating Molecule
regulatory device
A
B
C
D
E
F
G
H
Scar digestingdevice concentration builds
System Parts
List Of Parts
ScarTissue
Monoclonalantibody SMAD 3 DAXX HIPK2 ASK-1 MAP2k3
Checkpoint 1: Enzyme-Linked Immunosorbent Assay used to detect binding of monoclonal antibody to scar tissue
Checkpoint 2: Ligand Blot Overlay and Immunoprecipitation Assays are Used to monitor the binding of proteins to one another in the scar binding and healthy tissue binding cascades
Scar Binding Device
Parts Diagram (Left 1/3 of cell)
RBS
B0034
RBS
B0034
TT
B0015
Asp
Scar Digesting Sensor Device
Tar/envz
C0082
Tar/envz
C0082
E. coli binds to one end of monoclonal antibody
OmpRR0082
RBS
B0034
RBS
B0034
hk022CI
C0050
hk022CI
C0050
TT
B0015 pRR0050
HKCI
RBS
B0034
RBS
B0034
λ CI
C00051
λ CI
C00051
λCl
pBadI13453
RBS
B0034
RBS
B0034
hk022CI
C0051
hk022CI
C0051
TT
B0015
cI pRR0051
RBS
B0034
RBS
B0034
hk022CI
C0050
hk022CI
C0050
HKCIλCl
Trigger
MAP2k3
Parts Diagram (Middle 1/3 of cell)
Asp
To periostin secreting device
λ CI
C00051
λ CI
C00051
λCl
hk022CI
C0050
hk022CI
C0050
HKCI
TRIGGER
mOrange
E0030
mOrange
E0030
RBS
B0034
RBS
B0034
Checkpoint 2
Glow orange
collagenasecollagenaseRBS
B0034
RBS
B0034
Scar digesting device
Collagenase
AspA
C0083
AspA
C0083
RBS
B0034
RBS
B0034
Asp producer
AspA
Asp
ECFP
E0022
ECFP
E0022
RBS
B0034
RBS
B0034
Reporter 1
TT
B0015
Glow cyan
TT
B0015
periostinperiostinRBS
B0034
RBS
B0034
Heart regeneration device
periostin
GFP
J52028
GFP
J52028
RBS
B0034
RBS
B0034
Reporter 2
TT
B0015
Glow green
Parts Diagram (Right 1/3 of cell)
From scar binding device
pBadI13453
Our Highlighted Part: Periostin Secreting Device
•Original Gene Sequence taken from Human Chromosome #13•Coordinates: 13q13.3•Nucletiodes changed:
•1.EcoRI sites: 2148-2150 from GAA to GAG•‘2. PstI sites: 1305-1307 from CTG to CTA, 1572-1574 from GCT to GCA•3. SpeI sites: changed 1653-1655 from ACT to ACC•4. XbaI sites: changed 2496-2498 from TCT to TCC
•BioBrick Number BBa_1728950
Google.com
•For Protein secretion outside E.coli cell, attaching DNA construct
•Patent # 5223407•Inventors: Raymond Wong of Mississauga, CA and Margaret Sutherland of Cambridge, GB•Composed of:
•OmpA signal peptide•“Control Region”-lac promoter, tac promoter, and 5'AGGAGGAAAAAATT3' ribosome binding site.
Our Highlighted Part: Periostin Secreting Device (cont.)
Google.com
Unknowns
•What E. coli receptor proteins are best to use for binding to the monoclonal antibody?
•The protein kinases look good on paper; but will they function as predicted in vivo?
•Are we correct in assuming that we can synchronize the function of collagenase and periostin so that both can be secreted at the same time?
•Dosage and frequency of administration: how many injections are necessary, and how often must they be given?
Safety and Security Considerations
• It is necessary to prevent the immune system from attacking the E. coli in the bloodstream
Bactoblood chassis
Periostin is a growth factor, however similar clinical usage has not demonstrated carcinogenic side effects
•The E. coli bacteria will bind to the extracellular matrix of myocardial cells
• It may not be safe to block heart cell receptors by binding directly to them
google.com
Safety and Security Considerations (Continued)
It is necessary to ensure that collagenase only digests collagen in infarcted cells, not healthy cells
• If more accurate targeting is needed than our regulation device provides, a protein “tether” can be used
•Initial stages of the project can be performed in BL1 labs
•BL2 labs are necessary for subsequent development
The Competition: Who Else is Doing This?
• Our project was inspired by Gelfoam•A periostin-soaked gel applied directly to the heartOur product is less invasive
Gelfoam inserted into the heart (google.com)
Competition (Continued)
•Scar healing silicone sheets•Limited usage: must be applied daily, immediately after the wound has closed, and not longer than 3 months
•Low-energy laser irradiation
•Cost prohibitive
Scar-healing silicone sheet(google.com)
In Vivo Debugging: Fluorescent Markers
Glow orange Trigger “Off”
Glow cyan Scar Digesting Device “On”
Glow green
Heart Regeneration Devices “On”
Reporter Debugging Function
Testing and Debugging:
Mechanism 1:• Enzyme-Linked Immunosorbent Assay used to detect binding of monoclonal antibody to scar tissue
Mechanism 2:• Immunoprecipitation Assays• Method that uses the antigen-antibody reaction principle to identify a protein that reacts specifically with an antibody from mixture of proteins so that its quantity or physical characteristics can be examined.
6 Month Development Plan 2 Teams of 3 People Each
•Clone collagenase and express it in E. coli•Synthesize trigger mechanism for collagenase secretion
•Clone periostin and express it in E. coli•Synthesize trigger mechanism for periostin secretion
• Synthesize monoclonal antibody
GOAL: A successfully engineered plasmid ready for insertion into an E. coli bacterium
Thank You.
Are there any questions?
Referenceshttp://en.wikipedia.org/wiki/Monoclonal_antibodies
Information about monoclonal antibodies
http://www.ncbi.nlm.nih.gov/pubmed/16883602scar and healthy tissue binding
http://herkules.oulu.fi/isbn9514267214/html/x1329.htmlcardiac extracellular matrix
http://www.ncbi.nlm.nih.gov/pubmed/9521338 expression of cardiac proteins
http://www.ncbi.nlm.nih.gov/pubmed/10198196SMAD protein research
http://www.ingentaconnect.com/content/ap/mc/1999/00000031/00000003/art00902;jsessionid=cw3x1rheoz22.alice?format=print
SMAD and TGF -beta protein research
http://books.nap.edu/openbook.php?record_id=9450&page=8monoclonal antibodies
References (cont.)
http://content.nejm.org/cgi/content/full/344/23/1785engineered cardiomycotes, myocardial infarction
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T13-4CNCNXW-5&_user=501045&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000022659&_version=1&_urlVersion=0&_userid=501045&md5=b622660c2a4b7ab53b1f77
0f463c8979myocardial infarction description
http://www.medicalnewstoday.com/articles/92139.phpFibroblast/tissue buildup description
Wong, Raymond W. K. (Mississauga, CA), Sutherland, Margaret L. (Cambridge, GB) 1993. “Excretion of heterologous proteins from E. Coli.” United States
ALLELIX INC (CA). 5223407http://www.freepatentsonline.com/5223407.html
References (cont.)
http://www.wipo.int/pctdb/en/wo.jsp?ELEMENT_SET=F&LANGUAGE=ENG&KEY=05%2F019471&IA=05%2F019
471&DISPLAY=DESC periostin a natural response in mice to myocardial infarctions
http://www.nature.com/nrd/journal/v6/n9/full/nrd2406.html periostin experiment showing it improves heart function after
infarctions by regenerating tissue
http://www.nature.com/nm/journal/v13/n8/abs/nm1619.html article on prospects of periostin as treatment
http://www.childrenshospital.org/newsroom/Site1339/mainpageS1339P1sublevel319.html
article on how periostin was used to induce mature heart cells to grow